Development Of A LDH-ELISA For Detection Of Antibodies Against Mycoplasma Hyopneumoniae And Cloning Of The Ribonucleotide Reductase Gene Of Mycoplasma Hyopneumoniae

Mycoplasma hyopneumoniae is the etiological agent of Mycoplasma pneumoniae of swine (MPS), which is also named enzootic pneumoniae of swine( EPS), a chronic respiratory disease of swine. MPS is characterized by cough, sneezing, high morbidity rates, low mortality rates, retarded growth, poor food conversion. This pathogen colonizes the respiratory epithelia of swine and compromises their integrity through the induction of an inflammatory response. M. hyopneumoniae infection predispsees pigs to secondary invaders, such as Pasteurella multocida and Actinobacillus pleuropneumoniae, which exacerbate economic losses and may increase mortality. The DNA sequence of LDH encoding the early and specific immunogenic protein of M. hyopneumoniae has been determined. ELISA is widely used in the identification of animal diseases, because of its convenience, promptness, high specificity and sensitivity. Moreover, the cloning of the nrdF of Mycoplasma hyopneumoniae will be the groundworks of the development of the genetically engineering vaccine and it will facilitate the control of Mhp.Therefore, the researches are as follows. 1. Development of ELISA for antibody detection of Mycoplasma hyopneumoniaeThe molecular study revealed the species-specific property of LDH among Mhp and potential to elicit strong immune response. The truncated LDH gene was amplified from an attenuated Mycoplasma hypopneumoniae strain, ligated to pMD18-T vector for sequence analysis and to pGEX-KG for construction of recombinant expression plsamid which was transformed with E.coli BL21 competent cells. The protein was expressed in the form of solubility and insolubility and was recognized by positive reference serum by western blot. The soluble LDH protein was purified using Glutathione sepharose 4B bead and used as antigen to develop an indirect ELISA for detection of antibody against Mycoplasma hypopneumoniae. The cutoff value was determined by testing 69 IHA-negative sera. 92.5% of agreement was obtained between our new ELISA and commercial IHAkit by simultaneously detecting 120 samples. The good reproducibility and specificity were assessed. The assay was further applied to detect 671 clinically collected at random serum samples. The detection rates in sera from postweaning piglets, growing pigs, finishing pigs and adult breeding pigs were 44.3%, 3.0%, 17.44% and 73.41%, respectively, indicating the prevalence of Mycoplasma hypopneumoniae in pig herds that was consistent with previous reports.